Differential Infectivity of Original and Delta Variants of SARS-CoV-2 in Children Compared to Adults

ABSTRACT Although children of all ages are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, they have not been implicated as major drivers of transmission thus far. However, it is still unknown if this finding holds true with new variants of concern (VOC), such as Delta (B.1.617.2). This study aimed to examine differences in both viral RNA (as measured by cycle threshold [CT]) and viable-virus levels from children infected with Delta and those infected with original variants (OV). Furthermore, we aimed to compare the pediatric population infection trends to those in adults. We obtained 690 SARS-CoV-2 RT-PCR positive nasopharyngeal swabs from across Manitoba, Canada, which were further screened for mutations characteristic of VOC. Aliquots of sample were then provided for TCID50 (50% tissue culture infective dose) assays to determine infectious titers. Using a variety of statistical analyses we compared CT and infectivity of VOC in different age demographics. Comparing 122 Delta- to 175 OV-positive nasopharyngeal swab samples from children, we found that those infected with Delta are 2.7 times more likely to produce viable SARS-CoV-2 with higher titers (in TCID50 per milliliter), regardless of viral RNA levels. Moreover, comparing the pediatric samples to 130 OV- and 263 Delta-positive samples from adults, we found only that the Delta pediatric culture-positive samples had titers (TCID50 per milliliter) similar to those of culture-positive adult samples. IMPORTANCE These important findings show that children may play a larger role in viral transmission of Delta than for previously circulating SARS-CoV-2 variants. Additionally, they may suggest a mechanism for why Delta has evolved to be the predominant circulating variant.

than those infected with original variants. This manuscript has both epidemiological and virological importance but the manuscript requires some corrections before considering its publication. Areas for revision and correction: Please delete subheadings labelling numbers in the manuscript e.g. 1.0. Introduction Abstract: The abstract is concise and explains the study well. However to be in line with the journal style, replace the heading "Interpretation" with "Importance". Methods: SARS-CoV-2 RT-PCR For the variant mutations, they were mentioned "N501Y, 484K and 452R", please write them consistently. E.g E484K etc Statistical analysis: Page 6, line 180 -184: There should be citation of your previous work on this subject and citation of other studies where you mentioned about the delta variant infectious rate. In addition, what method was used to calculate the sample size, please elaborate on this "In our previous work, we found that adults had a culture positivity rate of 28.9%. Based on published data that the Delta variant may be 40-60% more infectious than wild type SARS-CoV-2, and assuming that this value would be related to culture positivity, we would require 116 pediatric samples to detect a 40% increase in culture positive rates, with a power of 0.  Figure 2: Comparison of Tissue Culture Infective dose 50% (TCID50) per mL by variant of concern (VOC) vs. age group. There was no difference in TCID50/ml between the different age categories with respect to the Delta variant specifically (P 0.68 Kruskal-Wallis ANOVA). In the pediatric age groups, the TCID50/ml was significantly higher for the Delta variant in the 11-17 age category than for OV (5620 [1780-17800] vs. 316 , P <0.001), but there was no difference in the 0-10 age category." Reviewer #3 (Comments for the Author): I would like to thank you for this wonderful work, which will be a qualitative addition to understanding important molecules of the COVID-19 pandemic, but there are some comments that should be reviewed.
In lines (239-243) It is preferable to delete this sentence. We still do not know what the side effects of vaccines are. Also, some studies have confirmed that there are side effects to the vaccine, and there are many opponents to the vaccine. Also, there are many voices that have not agreed to give vaccinations to children. The results of this study will tell us important things about how new mutations spread and how to stop them, and you don't need to mention the vaccine at the moment to children.
In References, You need to standardize the format for all references (style). There are some errors in the year of publication, as well as the contents of some references.

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Statistical Analysis 172
Statistical analysis were as previously described (20). Briefly, here we present normally 173 distributed data with means and standard deviations, and present non-normally distributed data 174 with medians and interquartile ranges (IQRs). We assessed normality using the Kolmorgorov-175 Smirnov test. We performed between-group comparisons using the Student t test or the Mann-176 Whitney test, and used the Fisher exact test for categorical data. We compared nonparametric 177 group medians using Kruskal-Wallis analysis of variance. We considered two-tailed p values 178 less than 0.05 as significant. We performed statistical analysis with Stata version 16.1 and 179 GraphPad Prism 9. In our previous work, we found that adults had a culture positivity rate of 180 28.9%. Based on published data that the Delta variant may be 40-60% more infectious than wild 181 type SARS-CoV-2, and assuming that this value would be related to culture positivity, we would 182 require 116 pediatric samples to detect a 40% increase in culture positive rates, with a power of 183 0.8 and α of 0.05 among children.  signifying that the quality of the samples is comparable between age groups. This also confirms 202 that there are no differential PCR inhibitors within the samples as tested and that all reagents and 203 protocols are working appropriately. 204 has been continuously scrutinized. Although earlier studies suggested that the spread of disease 218 from and amongst children is low, more recent evidence suggests that this may be changing 219 (15,21). As highly transmissible VOCs, such as Delta, arise globally, there is a renewed concern 220 regarding the pediatric populations role in seeding outbreaks and an increasing importance to The physiological mechanism for age discrepancies for SARS-CoV-2 infection is still 227 widely under investigation due to the potentially large number of biological, host and 228 environmental factors (23). These factors may include differences in the dynamics of shedding 9 from children as compared to adults and/or reflect differences in maturity of their respective 230 immune systems (24). 231 While it has been generally accepted that higher secondary attack rates are associated 232 with higher viral loads as measured by quantitative nucleic acid amplification methods (i.e. RT-

Limitations 249
There are limitations of this study that should be considered. This includes the lack of 250 clinical and epidemiological data linked to samples from this study. Incorporation of this data is 251 an important next step in establishing SARS-CoV2 Delta VOC transmission dynamics in 252 children. Additionally, without longitudinal sampling any difference in onset and duration of 253 viral shedding between Delta and OVs in children cannot be determined. The duration of 254 infectivity likely plays a major role in increased viral transmission of Delta compared to OVs 255 and should be further investigated. It is also possible that differences in RT-PCR and culturable 256 virus between children and adults are accounted for by the nature or quality of the swab sampling 257 differences between these demographics. However, the fact that the quantification of the house-258 keeping gene (BGB) from adults and children are comparable make this less likely (Figure 3). 259 Furthermore, the same samples are used for PCR testing and culturable virus and so if this is an 260 accepted critique, we should not consider similar Ct cut-offs in adults and children.

Reviewer 1:
Please delete subheadings labelling numbers in the manuscript e.g. 1.0. Introduction All numbering format for subheadings has been removed.
Abstract: The abstract is concise and explains the study well. However to be in line with the journal style, replace the heading "Interpretation" with "Importance".
The heading titled interpretation has been changed to Importance.

Methods: SARS-CoV-2 RT-PCR
For the variant mutations, they were mentioned "N501Y, 484K and 452R", please write them consistently. E.g E484K etc Thank you for pointing out this inconsistency, the single nucleotide polymorphism nomenclature has been updated on line 159 "N501Y, E484K and L452R" Statistical analysis: Page 6, line 180 -184: There should be citation of your previous work on this subject and citation of other studies where you mentioned about the delta variant infectious rate. In addition, what method was used to calculate the sample size, please elaborate on this "In our previous work, we found that adults had a culture positivity rate of 28.9%. Based on published data that the Delta variant may be 40-60% more infectious than wild type SARS-CoV-2, and assuming that this value would be related to culture positivity, we would require 116 pediatric samples to detect a 40% increase in culture positive rates, with a power of 0.8 and α of 0.05 among children. " We apologize for missing these references beforehand, the following references have been added as suggested: 21. Bullard   This grammatical error has been corrected, thank you.
In page 14, line 384: explain what these numbers are? " Figure 2: Comparison of Tissue Culture Infective dose 50% (TCID50) per mL by variant of concern (VOC) vs. age group. There was no difference in TCID50/ml between the different age categories with respect to the Delta variant specifically (P 0.68 Kruskal-Wallis ANOVA). In the pediatric age groups, the TCID50/ml was significantly higher for the Delta variant in the 11-17 age category than for OV (5620 [1780-17800] vs. 316 [178-2125], P <0.001), but there was no difference in the 0-10 age category." The numbers in figure 2 caption are the median TCID50/mL and the associated interquartile range for children (aged 11-17) infected with delta variant (5620[1780-17800]) and the original variant (316[178-2125]). As these numbers showed a significant difference we have also included the P value.
For clarity we have added a sentence to the statistical methods section Line 184 depicting how the statistical results are presented throughout the paper. " Statistical results are reported as (Median [Interquartile range], P value)."
The statistics on case and deaths has been updated as of July 15, 2022 on line 120. "Severe acute respiratory syndrome 2 (SARS-CoV-2), the etiological agent of COVID-19, has caused over 556 million cases and 6.3 million deaths globally as of July 15, 2022 (WHO Dashboard)." In lines (239-243) It is preferable to delete this sentence. We still do not know what the side effects of vaccines are. Also, some studies have confirmed that there are side effects to the vaccine, and there are many opponents to the vaccine. Also, there are many voices that have not agreed to give vaccinations to children. The results of this study will tell us important things about how new mutations spread and how to stop them, and you don't need to mention the vaccine at the moment to children.
The sentence has been deleted as suggested.
In References, You need to standardize the format for all references (style). There are some errors in the year of publication, as well as the contents of some references. Thank you for pointing this out. We have gone through the references and made it a consistent format as well as corrected the mistakes as follows: